Aging, atrophied skeletal muscles demonstrate a markedly impaired regenerative capacity when exposed to the same stress as young. This is a significant concern for veterans transitioning toward frailty or attempting to recover from joint surgery, as atrophy is exacerbated and these patients are unable to restore muscle mass to pre-surgery levels despite intensive physical efforts. The overarching goal of this project is to advance knowledge of the molecular mechanisms causing muscle regeneration impairment with increasing age. There is general consensus that the complex array of coordinated activities guiding developmental myogenesis- principally muscle progenitor (satellite cell, SC) recruitment and net muscle protein synthesis-is recapitulated during postnatal muscle regeneration. Identifying key mechanisms that disrupt these processes in aging muscle is essential to improve regenerative responses among old. Via human muscle genomic microarrays and network mapping, along with protein-level follow-up studies, we have identified an age-specific transcriptome response to mechanical stress-candidate signaling pathways and biological processes that were altered (at the transcript level) uniquely in the muscles of old (77 y) compared to young (34 y)-which drives our novel, central hypothesis that impaired muscle regeneration in old results from heightened resting muscle sensitivity to inflammatory mediators and consequent disruption of myogenic processes requisite for successful regeneration. Taking advantage of our tissue repository collected from 158 adults (90 women, 68 men) across four age groups (25, 35, 61, and 72 y) before and after a standardized mechanical stress, this hypothesis will be tested via the following series of aims focused on molecular and protein-level cell signaling studies in human muscle tissue specimens and primary SCs, and on human genomic microarrays. Specific Aim 1. We will determine the effects of age and sex on resting and mechanical stress-induced: cytokine signaling [NFkB, SAPK/JNK (TNF-1, IL-1), JAK/STAT3 (IL-6)];heat-shock protein content;ubiquitin- proteasome activity;translation initiation signaling;and pro-myogenic transcription factor expression in skeletal muscle. Specific Aim 2. We will determine the influence of donor age on the sensitivity of primary human SCs to inflammatory cytokines. Specific Aim 3: While our microarrays were revealing and sufficient to direct targeted Aims 1-2, they are preliminary (8 old, 8 young). We will therefore conduct additional genomic arrays to map the sex-specific, age-related onset and progression of changes in the transcriptome response to mechanical stress;identify other novel targets that may prove to be important mediators of age x sex differences. We fully expect this series of novel Aims to reveal causes of impairment as well as targets for potentiating muscle regeneration among old. PUBLIC HEALTH RELEVANCE: As veterans get older, two important changes in their skeletal muscles negatively affect their physical function and quality of life: 1) muscles get smaller (atrophy);and 2) muscles have a reduced ability to repair themselves after injury (impaired regenerative capacity). These are major concerns for aging veterans attempting to recover from injury or joint surgery, as these patients are unable to restore muscle mass to pre- surgery levels despite intensive physical efforts. The major goal of this research project is to determine key factors that cause muscle regeneration impairment as individuals get older. The project therefore holds an extremely high degree of scientific promise and is highly relevant to aging veterans.